Chengmin Zhang

Dynamic Compression Effects on Immature Nucleus Pulposus: a Study Using a Novel Intelligent and Mechanically Active Bioreactor.

Background: Previous cell culture and animal in vivo studies indicate the obvious effects of mechanical compression on disc cell biology. However, the effects of dynamic compression magnitude, frequency and duration on the immature nucleus pulposus (NP) from an organ-cultured disc are not well understood. Objective: To investigate the effects of a relatively wide range of compressive magnitudes, frequencies and durations on cell apoptosis and matrix composition within the immature NP using an intelligent and mechanically active bioreactor. Methods: Discs from the immature porcine were cultured in a mechanically active bioreactor for 7 days. The discs in various compressive magnitude groups (0.1, 0.2, 0.4, 0.8 and 1.3 MPa at a frequency of 1.0 Hz for 2 hours), frequency groups (0.1, 0.5, 1.0, 3.0 and 5.0 Hz at a magnitude of 0.4 MPa for 2 hours) and duration groups (1, 2, 4 and 8 hours at a magnitude of 0.4 MPa and frequency of 1.0 Hz) experienced dynamic compression once per day. Discs cultured without compression were used as controls. Immature NP samples were analyzed using the TUNEL assay, histological staining, glycosaminoglycan (GAG) content measurement, real-time PCR and collagen II immunohistochemical staining. Results: In the 1.3 MPa, 5.0 Hz and 8 hour groups, the immature NP showed a significantly increase in apoptotic cells, a catabolic gene expression profile with down-regulated matrix molecules and up-regulated matrix degradation enzymes, and decreased GAG content and collagen II deposition. In the other compressive magnitude, frequency and duration groups, the immature NP showed a healthier status regarding NP cell apoptosis, gene expression profile and matrix production. Conclusion: Cell apoptosis and matrix composition within the immature NP were compressive magnitude-, frequency- and duration-dependent. The relatively high compressive magnitude or frequency and long compressive duration are not helpful for maintaining the healthy status of an immature NP.

17beta-estradiol Attenuates TNF-α-Induced Premature Senescence of Nucleus Pulposus Cells through Regulating the ROS/NF-κB Pathway

Background: Accelerated cellular senescence within the nucleus pulposus (NP) region is a common feature of disc degeneration. Our previous work indicated that TNF-α promoted NP cell senescence. Although the intervertebral disc has been reported to be an estrogen-sensitive tissue, it is unclear whether estrogen can inhibit premature senescence of NP cells. Objective: To investigate whether 17beta-estradiol (E2) can attenuate TNF-α-induced premature senescence of NP cells and the potential mechanism behind this regulatory process. Methods: Isolated NP cells and intact intervertebral discs from healthy rats were cultured with or without TNF-α, E2 or their combination. The pan estrogen receptor (ER) antagonist ICI 182780 was used to investigate the role of ER. Direct and indirect indicators including cell proliferation, SA-β-Gal activity, telomerase activity, cell cycle, and the expression of matrix macromolecules (aggrecan and collagen II) and senescence markers (p16 and p53) were used to evaluate the premature senescence of NP cells. Additionally, intracellular reactive oxygen species (ROS) and NF-κB/p65 activity were also detected in the NP cell cultures. Results: In the NP cell cultures, E2 significantly increased cell proliferation potency, telomerase activity and the expression of matrix macromolecules but attenuated SA-β-Gal activity, senescence marker (p53 and p16) expression and G1 cycle arrest in TNF-α-treated NP cells. Furthermore, E2 inhibited ROS generation and phospho-NF-κB/p65 expression in the TNF-α-treated NP cells. However, the ER antagonist ICI 182780 abolished the effects of E2 on TNF-α-treated NP cells. In the disc organ cultures, E2 also significantly increased matrix synthesis, whereas it decreased senescence marker (p53 and p16) expression, which could be abolished by the ER antagonist ICI 182780. Conclusion: The interaction between E2 and ER can attenuate TNF-α-induced premature senescence of rat NP cells through interfering with the ROS/NF-κB pathway.

A Controlled Release Codelivery System of MSCs Encapsulated in Dextran/Gelatin Hydrogel with TGF-β3-Loaded Nanoparticles for Nucleus Pulposus Regeneration.

Mesenchymal stem cell- (MSC-) based therapy is regarded as a potential tissue engineering strategy to achieve nucleus pulposus (NP) regeneration for the treatment of intervertebral disc degeneration (IDD). However, it is still a challenge to induce MSC differentiation in NP-like cells when MSCs are implanted into the NP. The purpose of this study was to construct poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles as carriers for TGF-β3 controlled release and establish a codelivery system of a dextran/gelatin hydrogel with the nanoparticles for long-term processing of discogenesis differentiation. TGF-β3-loaded PLGA nanoparticles were prepared by the double-emulsion solvent evaporation method and seeded uniformly into the hydrogel. Morphological observations, an assessment of the release kinetics of TGF-β3, a cytotoxic assay, a cell proliferation test, a biochemical content assay, qRT-PCR, and immunohistological analyses of the codelivery system were conducted in the study. The results showed that the TGF-β3-loaded nanoparticles could release TGF-β3 gradually. The codelivery system exhibited favorable cytocompatibility, and the TGF-β3 that was released could induce MSCs to NP-like cells while promoting ECM-related biosynthesis. These results suggest this codelivery system may be employed as a promising carrier for discogenesis of MSCs in situ.

The inflammatory cytokine TNF-α promotes the premature senescence of rat nucleus pulposus cells via the PI3K/Akt signaling pathway

Premature senescence of nucleus pulposus (NP) cells and inflammation are two common features of degenerated discs. This study investigated the effects of the inflammatory cytokine TNF-α on the premature senescence of NP cells and the molecular mechanism behind this process. Rat NP cells were cultured with or without different concentrations of TNF-α for 1 and 3 days. The inhibitor LY294002 was used to determine the role of the PI3K/Akt pathway. NP cells that were incubated with TNF-α for 3 days followed by 3 days of recovery in the control medium were used to analyze cellular senescence. Results showed that TNF-α promoted premature senescence of NP cells, as indicated by decreased cell proliferation, decreased telomerase activity, increased SA-β-gal staining, the fraction of cells arrested in the G1 phase of the cell cycle, the attenuated ability to synthesize matrix proteins and the up-regulated expression of the senescence marker p16 and p53. Moreover, a high TNF-α concentration produced greater effects than a low TNF-α concentration on day 3 of the experiment. Further analysis indicated that the inhibition of the PI3K/Akt pathway attenuated the TNF-α-induced premature senescence of NP cells. Additionally, TNF-α-induced NP cell senescence did not recover after TNF-α was withdrawn. In conclusion, TNF-α promotes the premature senescence of NP cells, and activation of the PI3K/Akt pathway is involved in this process.

Role of the ERK1/2 pathway in osmolarity effects on nucleus pulposus cell apoptosis in a disc perfusion culture.

Osmolarity fluctuations are inevitable within the nucleus pulposus (NP). However, the effects of osmolarity on NP cell apoptosis within the organ‐cultured disc remain unclear. The objective of this study was to investigate effects of different osmolarity levels (hypo‐, iso‐, and hyper‐) and osmolarity modes (constant and cyclic) on NP cell apoptosis in a disc perfusion culture and to study the role of the ERK1/2 pathway in this regulatory process. Porcine discs were cultured for 7 days in different osmotic medium, including constant hypo‐, iso‐, and hyper‐osmolarity (330, 430, and 550 mOsm/L, respectively) and cyclic‐osmolarity (430 mOsm/L for 8 h, followed by 550 mOsm/L for 16 h). The role of the ERK1/2 pathway was investigated by using the pharmacological inhibitor U0126. NP cell apoptosis was analyzed by TUNEL staining, caspase‐3 activity, gene expression of Bcl‐2, Bax and caspase‐3 and protein expression of cleaved caspase‐3, and cleaved PARP. Our results showed that NP cell apoptosis was increased in hypo‐ and hyper‐osmolarity cultures compared to iso‐ or cyclic‐osmolarity culture, whereas the level of apoptosis in the iso‐osmolarity culture was lower than that in the cyclic‐osmolarity culture. When the ERK1/2 pathway was inhibited in the iso‐ and cyclic‐osmolarity cultures, the level of NP cell apoptosis was significantly increased. In conclusion, the effects of osmolarity on NP cell apoptosis depend on the osmolarity level (hypo‐, iso‐, or hyper‐) and osmolarity mode (constant or cyclic). Futhermore, inhibition of the ERK1/2 pathway promotes NP cell apoptosis in this process.

Estrogen Enhances Matrix Synthesis in Nucleus Pulposus Cell through the Estrogen Receptor β-p38 MAPK Pathway.

Background/Aims: Matrix homeostasis within the disc nucleus pulposus (NP) tissue is important for disc function. Increasing evidence indicates that sex hormone can influence the severity of disc degeneration. This study was aimed to study the role of 17β-estradiol (E2) in NP matrix synthesis and its underlying mechanism. Methods: Rat NP cells were cultured with (10-5, 10-7 and 10-9 M) or without (control) E2 for48 hours. The estrogen receptor (ER)-β antagonist PHTPP and ERβ agonist ERB 041 were used to investigate the role mediated by ERβ. The p38 MAPK inhibitor SB203580 was used to investigate the role of p38 MAPK signaling pathway. Gene and protein expression of SOX9, aggrecan and collagen II, glycosaminoglycan (GAG) content, and immunostaining assay for aggrecan and collagen II were analyzed to evaluate matrix production in rat NP cells. Results: E2 enhanced NP matrix synthesis in a concentration-dependent manner regarding gene and proetin expression of SOX9, aggrecan and collagen II, protein deposition of aggrecan and collagen II, and GAG content. Moreover, activation of p38 MAPK signaling pathway was increased with elevating E2 concentration. Further analysis indicated that ERB 041 and PHTPP could respectively enhance and suppress effects of E2 on matrix synthesis in NP cells, as well as activation of p38 MAPK pathway. Additionally, inhibition of p38 MAPK signaling pathway significantly abolished the effects of E2 on matrix synthesis. Conclusion: E2 can enhance matrix synthesis of NP cells and the ERβ/p38 MAPK pathway is involved in this regulatory process.

Matrix homeostasis within the immature annulus fibrosus depends on the frequency of dynamic compression: a study based on the self-developed mechanically active bioreactor

Evidence suggests that mechanical load is related to structural destruction of disk annulus fibrosus (AF) either in adult disk degeneration or in child disk acute injury. Both biochemical and biomechanical properties are different between immature and mature disks. However, the effects of mechanical compression on immature AF are not fully clear. This study was to investigate the effects of a relatively wide range of dynamic compressive frequency on matrix homeostasis within the immature AF. Immature disks from pig (3–4 months) were randomly assigned into the control group (non-compression) and compression groups (0.1, 0.5, 1.0, 3.0 and 5.0 Hz). All disks were bioreactor-cultured for 7 days. AF matrix production was evaluated by histology, gene expression, glycosaminoglycan (GAG) content, hydroxyproline (HYP) content and immunohistochemistry. Generally, no obvious difference was found in HE staining between control group and compression groups. However, alcian blue staining indicated proteoglycan content in the 5.0-Hz group was decreased compared with the control group and other compression groups. Similarly, a catabolic remodeling gene expression profile with the down-regulated matrix genes (aggrecan, collagen I and collagen II) and tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-3) and the up-regulated matrix catabolic enzymes (ADAMTS-4 and MMP-3) was found in the 5.0-Hz group. Further analysis indicated that GAG content, HYP content and aggrecan protein deposition were also decreased in the 5.0-Hz group. Hence, we concluded that matrix homeostasis within the immature AF was compressive frequency dependent, and the relatively higher frequency (5.0 Hz) is unfavorable for matrix production within the immature AF. These findings will contribute to further understanding of the relationship between mechanical compression and immature AF biosynthesis.

N-Cadherin-Mediated Activation of PI3K/Akt-GSK-3β Signaling Attenuates Nucleus Pulposus Cell Apoptosis Under High-Magnitude Compression

Background/Aims: Mechanical overloading-induced nucleus pulposus (NP) apoptosis plays an important role in the pathogenesis of intervertebral disc degeneration. N-cadherin (N-CDH)-mediated signaling preserves normal NP cell phenotype. This study aims to investigate the effects of N-CDH on NP cell apoptosis under high-magnitude compression and the underlying mechanism behind this process. Methods: Rat NP cells seeded on scaffold were perfusion-cultured using a self-developed perfusion bioreactor for 5 days and experienced different magnitudes (2% and 20% compressive deformation, respectively) of compression at a frequency of 1.0 Hz for 4 hours once per day. The un-loaded NP cells were used as controls. Lentivirus-mediated N-CDH overexpression and inhibitor LY294002 were used to further investigate the role of N-CDH and PI3K/Akt pathway under high-magnitude compression, respectively. NP cell apoptosis was evaluated by caspase-3 activity measured using a commercial kit, flow cytometry, and expression of apoptosis-related molecules analyzed by real-time PCR and western blotting assays. Results: High-magnitude compression significantly increased apoptotic NP cells, caspase-3 activity and expression of pro-apoptotic molecules (Bax and caspase-3/cleaved caspase-3), but decreased expression of anti-apoptotic molecule (Bcl-2). High-magnitude compression decreased expression of N-CDH, p-Akt and p-GSK-3β. However, N-CDH overexpression attenuated NP cell apoptosis and increased expression of p-Akt and p-GSK-3β under high-magnitude compression. Further analysis showed that inhibition of the PI3K/Akt pathway suppressed NP cell apoptosis and decreased expression of p-GSK-3β, but had no significant effects on N-CDH expression under high-magnitude compression. Conclusion: N-CDH can attenuate NP cell apoptosis through activating the PI3K/Akt-GSK-3β signaling under high-magnitude compression.

Improved Monosegment Pedicle Instrumentation for Treatment of Thoracolumbar Incomplete Burst Fractures

Aim. Comparing the clinical results of improved monosegment pedicle instrumentation (iMSPI) and short-segment pedicle instrumentation (SSPI) retrospectively. Method. 63 patients with thoracolumbar incomplete burst fracture were managed with iMSPI or SSPI. 30 patients were managed with iMSPI and fusion. 33 patients were managed with SSPI and fusion. Operative time, blood loss, postoperative drainage, and complications were recorded. Percentage of anterior body height compression (ABHC%) and sagittal index (SI) were obtained preoperatively, one week postoperatively, and at the last followup. Results. The blood loss and postoperative drainage were significantly less in the iMSPI group than in SSPI group (P < 0.05). The follow-up duration of the two groups was not significantly different (P > 0.05). At 12 months postoperatively posterolateral fusion was obtained satisfactorily. Neither preoperative ABHC% and SI nor postoperative SI were significantly different (P > 0.05), but there was a significant difference in postoperative ABHC% (P = 0.000). The ABHC% and SI were not significantly different between the two groups at the last followup (P > 0.05). There were no fixation failures or other complications. Summary. IMSPI yielded satisfactory results similar to those of SSPI in patients with type A3.1/3.2 thoracolumbar fractures. IMSPI is recommended for minor trauma, reducing one-segment fusion, and maximization of the remaining motor function.

The influence of L4–S1 Dynesys® dynamic stabilization versus fusion on lumbar motion and its relationship with lumbar degeneration: a retrospective study

BackgroundThe aim of this study is to evaluate the efficacy of Dynesys® posterior dynamic stabilization (PDS) in the treatment of L4–S1 degenerative diseases and to assess the influence of postoperative motion on lumbar degeneration.MethodsIncluded in this retrospective study were patients with L4–S1 degenerative disease who underwent fusion or PDS from September 2010 to September 2014. Clinical outcomes were assessed by preoperative and postoperative visual analog scale (VAS) and Oswestry Disability Index (ODI). Preoperative and postoperative X-rays assessed range of motion (ROM) of the non-surgical and surgical levels and whole lumbar. MRI assessed degeneration of non-surgical levels.ResultsA total of 56 consecutive patients were divided into two groups: group A, PDS, and group B, fusion. Patient demographics and baseline characteristics were similar in the two groups. In both groups, there was a significant difference between preoperative and postoperative VAS and ODI scores (P < 0.05). However, there was a significant difference in a 6-month follow-up ODI between the two groups (P < 0.05). X-rays showed PDS patients partially maintained surgical level ROM and non-surgical level ROM increased less than in the fusion group. MRI showed adjacent segment degeneration (ASD) in both groups, and patients whose preoperative L3–4 Pfirrmann classification was higher than grade 2 had more ASD than lower than grade 2.ConclusionPDS can maintain surgical level ROM and had less influence on whole and non-surgical level ROM. Following PDS, patients recovered faster and had a better lumbar function. It may be a better choice for multi-level lumbar degenerative diseases.